The overall objective for these studies is to gain insight on the molecular events that occur at the single cell level when natural killer (NK) cells interact with cytolytically susceptible and non-susceptible target cells. The intercellular contact area, called the immune synapse (IS), undergoes temporal and spatial changes that are thought to be of critical importance for optimizing the cellular interactions and control the outcome. Most studies of the IS have been performed with helper T-cells interacting with antigen-presenting cells (APC). Formation of the helper T-cell/APC IS (ThIS) demonstrates a tightly regulated process involving sequential recruitment of signaling molecules to compartmentalized areas within the intercellular contact site. The long-term goal for our proposed studies is to test the hypothesis that the IS formed during cytolytic NK cell interactions with target cells (NKIS) display important differences from the ThIS that develops during initiation of clonal T cell proliferation. We will in the present proposal utilize the MHC class I regulated NK cell cytotoxicity model for analysis of the cytolytic and non-cytolytic NKIS. We demonstrate in our preliminary results that these events are associated with the formation of both a cytolytic NKIS and a non-cytolytic NKIS. We hypothesize that a distinct subset of signal transduction molecules will be recruited to the cytolytic NKIS. Specifically, we expect to identify differential recruitment of signaling molecules that mediate granule exocytosis while enzymes and adaptor molecules engaged in cell proliferation will be absent from the synapse or displaced away from active signaling components. We also hypothesize that the inhibitory NK receptors with ligand specificity for MHC class I molecules regulate the signaling pathways by ligand-induced movements in the synaptic region. Finally, we hypothesize that polymerization of actin-cytoskeleton mediated by the Wiskott-Aldrich Syndrome Protein (WASP) is not an essential component in mediating the cytotoxic effector events by NK cells. The proposal has three specific aims: (1) To characterize the MHC class I regulated temporal and spatial organization of signal transduction molecules in the synaptic region of NK cells in non-cytolytic and cytolytic interactions with target cells. (2) To characterize the temporal and spatial organization of NK receptors and their HLA ligands in the contact region of NK cell-target cell conjugates in cytolytic and non-cytolytic combinations. (3) To characterize the temporal and spatial organization of the cytolytic and non-cytolytic NKIS in patients with different mutations in the WASP gene. We expect these studies to provide new insight on how NK cells interact with target cells and how MHC class I molecules regulate these processes.